In the yeast, Saccharomyces cerevisiae, the silent mating loci, HMLalpha and HMRa, are permanently inactivated in normal cells. This repression is part of the mechanism which generates different cell types (a, alpha, diploid) and allows mating to occur. Silencing occurs between specific DNA boundaries, termed 'silencers', and requires the function of at least the four proteins Sir1, Sir2, Sir3 and Sir4. Recently, we found that deletions in the extremely conserved histone H4 N-terminus of yeast (but not similar deletions in the N-termini of histones H2A or H2B) activate the silent mating loci specifically. We propose here experiments which will help uncover many of the details in the mechanism of chromosomal repression of the silent mating loci. Using directed mutagenesis we intend to determine a) which sequences in histone H4 are required for silencing and whether there is special significance to H4 N-terminal acetylation in modifying repression of the silent mating loci. b) We will determine whether histones are involved in other mating locus specific functions such as gene conversion, autonomous replication and segregation. c) Antibodies to Sir proteins will be used to determine whether these repressor proteins interact directly with silencer chromatin isolated as episomes and in particular whether they interact with the H4 N-terminus. d) We will ask whether there are other, as yet undefined factors which interact with H4 to repress the silent mating loci. This will be done by the identification of genes whose products suppress histone gene mutations. e) We will determine whether it is H4 alone or the H3- H4 complex which is involved in repression of HMLalpha and HMRa. The chromosomal repression of the silent mating loci in yeast has analogies to the inactivation of single copy genes seen in heterochromatin of flies and mammals. This too is accompanied by changes in chromatin structure and results in long-term genetic inactivation. The understanding of the molecular basis of silencing, is likely to have a profound effect on our understanding of terminal repression, and therefore cellular differentiation, in all eukaryotes. This knowledge should impact our ability to deal with major illness, such as cancer, in which cellular differentiation is abnormal.